Impact of multi-metals (Cd, Pb and Zn) exposure on the physiology of the yeast Pichia kudriavzevii

Metal contamination of the environment is frequently associated to the presence of two or more metals. This work aimed to study the impact of a mixture of metals (Cd, Pb and Zn) on the physiology of the non-conventional yeast Pichia kudriavzevii. The incubation of yeast cells with 5 mg/l Cd, 10 mg/l Pb and 5 mg/l Zn, for 6 h, induced a loss of metabolic activity (assessed by FUN-1 staining) and proliferation capacity (evaluated by a clonogenic assay), with a small loss of membrane integrity (measured by trypan blue exclusion assay). The staining of yeast cells with calcofluor white revealed that no modification of chitin deposition pattern occurred during the exposure to metal mixture. Extending for 24 h, the exposure of yeast cells to metal mixture provoked a loss of membrane integrity, which was accompanied by the leakage of intracellular components. A marked loss of the metabolic activity and the loss of proliferation capacity were also observed. The analysis of the impact of a single metal has shown that, under the conditions studied, Pb was the metal responsible for the toxic effect observed in the metal mixture. Intracellular accumulation of Pb seems to be correlated with the metals' toxic effects observed.

SOIL CONTAMINATION IN PUBLIC SQUARES IN BELO HORIZONTE, MINAS GERAIS, BY CANINE PARASITES IN DIFFERENT DEVELOPMENTAL STAGES

SUMMARY To evaluate soil contamination by parasites in different developmental stages in public squares used as recreation and leisure areas for children in Belo Horizonte (MG, Brazil), 210 soil samples and 141 canine fecal samples were collected from 42 squares in the city. These samples were analyzed by the Caldwell and Caldwell technique and the Hoffman, Pons, and Janer technique. Of the samples analyzed, 89 (42.4%) soil samples and 104 (73.5%) fecal samples were contaminated with Ancylostoma sp., Toxocara sp., Trichuris sp., or Dipylidium sp. eggs; Giardia sp. cysts; or Isospora sp. oocysts. The commonest parasite was Ancylostoma sp., found in 85% soil and 99% fecal samples, followed by Toxocara sp., found in 43.7% soil and 30.7% fecal samples.

OCCUPATIONAL EXPOSURES TO BODY FLUIDS AND BEHAVIORS REGARDING THEIR PREVENTION AND POST-EXPOSURE AMONG MEDICAL AND NURSING STUDENTS AT A BRAZILIAN PUBLIC UNIVERSITY

A cross-sectional study was conducted to assess the frequencies and characteristics of occupational exposures among medical and nursing students at a Brazilian public university, in addition to their prevention and post-exposure behavior. During the second semester of 2010, a self-administered semi-structured questionnaire was completed by 253/320 (79.1%) medical students of the clinical course and 149/200 (74.5%) nursing students who were already performing practical activities. Among medical students, 53 (20.9%) suffered 73 injuries, which mainly occurred while performing extra-curricular activities (32.9%), with cutting and piercing objects (56.2%), in the emergency room (39.7%), and as a result of lack of technical preparation or distraction (54.8%). Among nursing students, 27 (18.1%) suffered 37 injuries, which mainly occurred with hollow needles (67.6%) in the operating room or wards (72.2%), and as a result of lack of technical preparation or distraction (62.1%). Among medical and nursing students, respectively, 96.4% and 48% were dissatisfied with the instructions on previously received exposure prevention; 48% and 18% did not always use personal protective equipment; 67.6% and 16.8% recapped used needles; 49.3% and 35.1% did not bother to find out the source patient's serological results post-exposure; and 1.4% and 18.9% officially reported injuries. In conclusion, this study found high frequencies of exposures among the assessed students, inadequate practices in prevention and post-exposure, and, consequently, the need for training in “standard precautions” to prevent such exposures.

Treatment of a nineteenth century male portrait in oil including the characterisation of materials, technique and a study of the lead soap aggregation in the paint composite

Dissertação para obtenção do Grau de Mestre em Conservação e Restauro

Human immune responses during schistosomiasis mansoni

Studies of immune responses as they occur in patients with schistosomiasis appear to progress relative to corrent technological advances, and to advance despite the understandable inability to pursue in vivo manipulations in this host/parasite system. Emphasis is most often placed on making immunological comparisons between such patient groups as reinfected/non-reinfected, intestinals/hepatosplenic, high/low intensities of infection, infected/uninfected within endemic areas, and those born of infected/uninfected mothers. Based on these types of comparisons, reasonable conjectures can be made regarding the immunological occurrences during this chronic exposure condition. Some consideration is now being given to the immune mechanisms of some of the observations made, and while some of these must then be carried back to experimental models for further manipulation-based analysis, new technological developments continue to assist in the field/bench ability to ask questions that might assist our understanding to a point where this knowledge can be applied to shaping developmental approaches to vaccine development and the goal of alleviating morbidity.

Mercury exposure and malaria prevalence among gold miners in Pará, Brazil

Economic development, including resource extraction, can cause toxic exposures that interact with endemic infectious diseases. Mercury is an immunotoxic metal used in the amalgamation of gold, resulting in both occupational exposures and environmental pollution. A cross-sectional medical survey was conducted in 1997 on 135 garimpeiros in Para, Brazil, because of their risks of both mercury exposure and malaria transmission. Mean levels of blood and urine mercury were well above non-exposed background levels. Twenty-six subjects had malaria parasitemia: Health symptoms consistent with mercury exposure were reported, but neither symptoms nor signs correlated with mercury levels in blood or urine. We did not find a dose response relationship between mercury exposure and likelihood of prevalent malaria infection, but there was a possible reduction in acquisition of immunity that may be associated with conditions in gold mining, including mercury exposure.

Serological screening and toxoplasmosis exposure factors among pregnant women in South of Brazil

Serological screening and evaluation of exposure factors for Toxoplasma gondii transmission were conducted in 2126 pregnant women from southern Brazil. Specific antibodies against Toxoplasma gondii were presented by 74.5% (n=1583) of the pregnant women evaluated. Contact with soil was found to be the major factor for infection.

Human exposure to potential rabies virus transmitters in Olinda, State of Pernambuco, between 2002 and 2006

The aim of the present study was to evaluate the data on human exposure to potential rabies virus transmitters in Olinda, State of Pernambuco, Brazil. Data from 7,062 patients who underwent antirabies prophylactic treatment in Olinda between 2002 and 2006 were analyzed. As expected, dogs and cats were involved in most of the cases; i.e. 82.3 and 16.3%, respectively. Attacks by nonhuman primates, bats and other species (unspecified) were also reported. Among the 7,062 patients who underwent antirabies treatment, 582 patients abandoned the treatment, either by indication from the health unit (195) or by their own decision (387). In conclusion, this study has indicated that prophylaxis for human rabies in this urban area will require a multifaceted approach, including health education, post-exposure prophylaxis, systematic vaccination for dogs and cats, and possibly selective control over wild animals such as hematophagous bats.

The role of Grainy head in epithelial tissue growth

Part of the results of this thesis was presented in the following meetings: Susana Ponte, Lara Carvalho, Inês Cristo and António Jacinto. The role of Grainy head in epithelial tissue growth. Drostuga 2013. Faro, Portugal, January 3rd 2014 [poster] Susana Ponte, Lara Carvalho, Inês Cristo and António Jacinto. The role of Grainy head in epithelial tissue growth. Drostuga 2014. Tomar, Portugal, September 5th-6th 2014 [poster]

Morphological diversification through the evolution of developmental hierarchies

Changes in development impact the final form of organisms and compose the natural variation that is the raw material for evolution. Development is hierarchically structured in progressive series of cell fate determination and differentiation. How does variation in different stages of development contribute to morphological diversification?

Gold nanoparticles for the detection of DNA adducts as biomarkers of exposure to acrylamide

The main objective of this thesis was the development of a gold nanoparticle-based methodology for detection of DNA adducts as biomarkers, to try and overcome existing drawbacks in currently employed techniques. For this objective to be achieved, the experimental work was divided in three components: sample preparation, method of detection and development of a model for exposure to acrylamide. Different techniques were employed and combined for de-complexation and purification of DNA samples (including ultrasonic energy, nuclease digestion and chromatography), resulting in a complete protocol for sample treatment, prior to detection. The detection of alkylated nucleotides using gold nanoparticles was performed by two distinct methodologies: mass spectrometry and colorimetric detection. In mass spectrometry, gold nanoparticles were employed for laser desorption/ionisation instead of the organic matrix. Identification of nucleotides was possible by fingerprint, however no specific mass signals were denoted when using gold nanoparticles to analyse biological samples. An alternate method using the colorimetric properties of gold nanoparticles was employed for detection. This method inspired in the non-cross-linking assay allowed the identification of glycidamide-guanine adducts and DNA adducts generated in vitro. For the development of a model of exposure, two different aquatic organisms were studies: a goldfish and a mussel. Organisms were exposed to waterborne acrylamide, after which mortality was recorded and effect concentrations were estimated. In goldfish, both genotoxicity and metabolic alterations were assessed and revealed dose-effect relationships of acrylamide. Histopathological alterations were verified primarily in pancreatic cells, but also in hepatocytes. Mussels showed higher effect concentrations than goldfish. Biomarkers of oxidative stress, biotransformation and neurotoxicity were analysed after prolonged exposure, showing mild oxidative stress in mussel cells, and induction of enzymes involved in detoxification of oxygen radicals. A qualitative histopathological screening revealed gonadotoxicity in female mussels, which may present some risk to population equilibrium.

Distribution of hepatitis C virus genotypes among different exposure categories in the State of Pará, Brazilian Amazon

INTRODUCTION: Epidemiological studies concerning HCV genotypic distribution in the Brazilian Amazon are scarce. Thus, this study determined the patterns of distribution of HCV genotypes among different exposure categories in the State of Pará, Brazilian Amazon. METHODS: A cross-sectional study was conducted on 312 HCV-infected individuals belonging to different categories of exposure, who were attended at the HEMOPA, CENPREN and a private hemodialysis clinic in Belém. They were tested for HCV antibodies using an immunoenzymatic test, RNA-HCV, using real-time PCR and HCV genotyping through phylogenetic analysis of the 5' UTR. The population groups were epidemiologically characterized according to data collected in a brief interview or medical consultation. RESULTS: Genotype 1 predominated in all the different categories of HCV exposure. HCV genotypic distribution among blood donors comprised genotypes 1 (94%) and 3 (6%). All patients with chronic hematologic diseases had HCV genotype 1. The genotypic distribution in illicit-drug users comprised genotypes 1 (59.6%) and 3 (40.4%). In patients under hemodialysis, genotypes 1 (90.1%), 2 (3.3%), and 3 (6.6%) were detected. Finally, the frequency of genotypes 1 and 3 was significantly different between the groups: BD and DU, PUH and DU, PUH and PCHD and PCHD and DU. CONCLUSIONS: The genotypic frequency and distribution of HCV in different categories of exposure in the State of Pará showed a predominance of genotype 1, regardless of the possible risk of infection.

Cellular reprogramming strategies for degenerative disorders involving the retinal pigment epithelium

RESUMO: A reprogramação celular permite que uma célula somática seja reprogramada para outra célula diferente através da expressão forçada de factores de transcrição (FTs) específicos de determinada linhagem celular, e constitui uma área de investigação emergente nos últimos anos. As células somáticas podem ser experimentalmente manipuladas de modo a obter células estaminais pluripotentes induzidas (CEPi), ou convertidas directamente noutro tipo de célula somática. Estas descobertas inovadoras oferecem oportunidades promissoras para o desenvolvimento de novas terapias de substituição celular e modelos de doença, funcionando também como ferramentas valiosas para o estudo dos mecanismos moleculares que estabelecem a identidade celular e regulam os processos de desenvolvimento. Existem várias doenças degenerativas hereditárias e adquiridas da retina que causam deficiência visual devido a uma disfunção no tecido de suporte da retina, o epitélio pigmentar da retina (EPR). Uma destas doenças é a Coroideremia (CHM), uma doença hereditária monogénica ligada ao cromossoma X causada por mutações que implicam a perda de função duma proteína com funções importantes na regulação do tráfico intracelular. A CHM é caracterizada pela degenerescência progressiva do EPR, assim como dos foto-receptores e da coróide. Resultados experimentais sugerem que o EPR desempenha um papel importante na patogénese da CHM, o que parece indicar uma possível vantagem terapêutica na substituição do EPR nos doentes com CHM. Por outro lado, existe uma lacuna em termos de modelos in vitro de EPR para estudar a CHM, o que pode explicar o ainda desconhecimento dos mecanismos moleculares que explicam a patogénese desta doença. Assim, este trabalho focou-se principalmente na exploração das potencialidades das técnicas de reprogramação celular no contexto das doenças de degenerescência da retina, em particular no caso da CHM. Células de murganho de estirpe selvagem, bem como células derivadas de um ratinho modelo de knockout condicional de Chm, foram convertidos com sucesso em CEPi recorrendo a um sistema lentiviral induzido que permite a expressão forçada dos 4 factores clássicos de reprogramação, a saber Oct4, Sox2, Klf4 e c-Myc. Estas células mostraram ter equivalência morfológica, molecular e funcional a células estaminais embrionárias (CES). As CEPi obtidas foram seguidamente submetidas a protocolos de diferenciação com o objectivo final de obter células do EPR. Os resultados promissores obtidos revelam a possibilidade de gerar um valioso modelo de EPR-CHM para estudos in vitro. Em alternativa, a conversão directa de linhagens partindo de fibroblastos para obter células do EPR foi também abordada. Uma vasta gama de ferramentas moleculares foi gerada de modo a implementar uma estratégia mediada por FTs-chave, seleccionados devido ao seu papel fundamental no desenvolvimento embrionário e especificação do EPR. Conjuntos de 10 ou menos FTs foram usados para transduzir fibroblastos, que adquiriram morfologia pigmentada e expressão de alguns marcadores específicos do EPR. Adicionalmente, observou-se a activação de regiões promotoras de genes específicos de EPR, indicando que a identidade transcricional das células foi alterada no sentido pretendido. Em conclusão, avanços significativos foram atingidos no sentido da implementação de tecnologias de reprogramação celular já estabelecidas, bem como na concepção de novas estratégias inovadoras. Metodologias de reprogramação, quer para pluripotência, quer via conversão directa, foram aplicadas com o objectivo final de gerar células do EPR. O trabalho aqui descrito abre novos caminhos para o estabelecimento de terapias de substituição celular e, de uma maneira mais directa, levanta a possibilidade de modelar doenças degenerativas da retina com disfunção do EPR numa placa de petri, em particular no caso da CHM.---------------ABSTRACT: Cellular reprogramming is an emerging research field in which a somatic cell is reprogrammed into a different cell type by forcing the expression of lineage-specific transcription factors (TFs). Cellular identities can be manipulated using experimental techniques with the attainment of pluripotency properties and the generation of induced Pluripotent Stem (iPS) cells, or the direct conversion of one somatic cell into another somatic cell type. These pioneering discoveries offer new unprecedented opportunities for the establishment of novel cell-based therapies and disease models, as well as serving as valuable tools for the study of molecular mechanisms governing cell fate establishment and developmental processes. Several retinal degenerative disorders, inherited and acquired, lead to visual impairment due to an underlying dysfunction of the support cells of the retina, the retinal pigment epithelium (RPE). Choroideremia (CHM), an X-linked monogenic disease caused by a loss of function mutation in a key regulator of intracellular trafficking, is characterized by a progressive degeneration of the RPE and other components of the retina, such as the photoreceptors and the choroid. Evidence suggest that RPE plays an important role in CHM pathogenesis, thus implying that regenerative approaches aiming at rescuing RPE function may be of great benefit for CHM patients. Additionally, lack of appropriate in vitro models has contributed to the still poorly-characterized molecular events in the base of CHM degenerative process. Therefore, the main focus of this work was to explore the potential applications of cellular reprogramming technology in the context of RPE-related retinal degenerations. The generation of mouse iPS cells was established and optimized using an inducible lentiviral system to force the expression of the classic set of TFs, namely Oct4, Sox2, Klf4 and c-Myc. Wild-type cells, as well as cells derived from a conditional knockout (KO) mouse model of Chm, were successfully converted into a pluripotent state, that displayed morphology, molecular and functional equivalence to Embryonic Stem (ES) cells. Generated iPS cells were then subjected to differentiation protocols towards the attainment of a RPE cell fate, with promising results highlighting the possibility of generating a valuable Chm-RPE in vitro model. In alternative, direct lineage conversion of fibroblasts into RPE-like cells was also tackled. A TF-mediated approach was implemented after the generation of a panoply of molecular tools needed for such studies. After transduction with pools of 10 or less TFs, selected for their key role on RPE developmental process and specification, fibroblasts acquired a pigmented morphology and expression of some RPE-specific markers. Additionally, promoter regions of RPE-specific genes were activated indicating that the transcriptional identity of the cells was being altered into the pursued cell fate. In conclusion, highly significant progress was made towards the implementation of already established cellular reprogramming technologies, as well as the designing of new innovative ones. Reprogramming into pluripotency and lineage conversion methodologies were applied to ultimately generate RPE cells. These studies open new avenues for the establishment of cell replacement therapies and, more straightforwardly,raise the possibility of modelling retinal degenerations with underlying RPE defects in apetri dish, particularly CHM.

Ultrastructural study on the morphological changes to male worms of Schistosoma mansoni after in vitro exposure to allicin

INTRODUCTION: Garlic has a wide range of actions, including antibacterial, antiviral, antifungal, antiprotozoal and anthelmintic actions. This antiparasitic activity has been attributed to allicin, which is the main constituent of garlic. The present study aimed to investigate the in vitro activity of allicin on the tegument of adult Schistosoma mansoni worms using scanning electron microscopy. METHODS: Swiss Webster mice were infected with S. mansoni cercariae (100 per mouse) and sacrificed 50 days later to acquire the adult worms. These worms were collected by perfusion and placed in RPMI medium 1,640 at 37°C before transferring to RPMI media containing 0 (control), 5, 10, 15 and 20mg/mL of allicin, where they were incubated for 2h. The worms were fixed in 2.5% glutaraldehyde solution, washed twice, post-fixed in osmium tetroxide, washed twice and then dehydrated with ascending grades of ethanol. The samples were air-dried, mounted on stubs, gold coated in an ion sputtering unit and viewed using a scanning electron microscope. RESULTS: A concentration of 5mg/mL caused wrinkling in the tegument; a concentration of 10mg/mL resulted in changes to tubercles and loss or modification of spines. With 15 and 20mg/mL increasing damage to the tegument could be seen, such as vesicle formation and the presence of ulcers. CONCLUSIONS: These findings demonstrate the effect of allicin on adult S. mansoni worms and indicate that most of the changes occur at concentrations greater than that normally indicated for treatment.

Managing talent through developmental job experiences (DJE): The mediating role of DJE between HPWS and employees' outcomes

In the competitive landscape of the 21st century, effectively managing human capital in firms is considered to be a potential source of sustainable performance. Therefore, in this study, we tested the influence of high-performance work systems, as a talent management tool, on employees’ experience of developmental jobs. Then, we tested the mediating effect of such experiences on employees’ engagement, exhaustion, performance and turnover intention. With a sample of 254 employees of a diversity of companies and sectors of activity, our findings demonstrated that high-performance practices increase engagement, via the promotion of developmental experiences of fit, which improves performance and decreases turnover intention. Besides, those practices do not control for the pressure dimension of the developmental job experiences that increases exhaustion and turnover intention despite not worsening performance.